This invention pertains to the field of detecting intermolecular interactions in vivo and in vitro using chimeric endonucleases that include a nuclease cleavage domain that is linked to a moiety that is capable of directly or indirectly binding to a molecule of interest. The provided chimeric endonucleases are useful for determining the location at which a protein becomes associated with a nucleic acid or other molecule.
Molecular interactions form the basis of many biological and chemical events. These include enzymatic reactions, hormone-ligand interactions, drug or toxin-protein interaction and other protein-protein, protein-nucleic acid, and nucleic acid-nucleic acid interactions. Therefore, in order to understand these biological and chemical events, the ability to detect contact or close proximity between two known molecules would be of great value.
The techniques to visualize a protein complex formed on a particular nucleic acids sequence, such as the electrophoretic mobility shift assay (EMSA) and footprinting assays, in vitro and in vivo, have been crucial to understanding how transcription occurs. Present techniques, however, have serious limitations. In a living cell, transcription happens in a far more complex environment than can be duplicated in vitro and as a result, in vitro techniques will not always depict accurately the situation in vivo. For example, in vitro techniques have not been useful in studying long range interactions ( greater than 1 kb) such as that of the xcex2-globin LCR which play an important role in transcription in a living cell. In vivo footprinting, on the other hand, reflects protein-DNA interactions in a living cell, but the identity of the complex creating the footprint is not known.
Hybrid affinity cleaving proteins composed of a DNA binding domain of a protein and a Fok 1 endonuclease have been used in vitro for cleavage of nucleic acids in a variation of in vitro footprinting methods. See, Chandrasegaran, U.S. Pat. Nos. 5,487,994 and 5,436,150; Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160 and Kim et al. Proc. Natl. Acad. Sci. USA (1994) 91: 883-887. However, here again, it has typically been assumed that the DNA binding domain is sufficient to confer sequence specific binding, and therefore only the DNA binding domain of the transcription factor has been included in the hybrid protein. In vitro, this assumption may be correct, since there are no other proteins present when the hybrid protein binds to the DNA. However, this assumption may not apply in vivo, and may not provide a realistic assessment of the nature of an in vivo DNA-protein complex. Furthermore, these methods do not provide any way of assessing the interaction of proteins which bind indirectly to a nucleic acid, such as proteins which form a transcription initiation complex.
Previously available methods of detecting interactions between molecules in the context of cells or organisms have been hampered by a lack of resolution and insufficient sensitivity. With the current state of the art technique, which combines confocal microscopy and immunofluorescence, two molecular species can be visualized simultaneously. If the two molecular species interact, their fluorescent signals should colocalize. However, because the limit of the resolution for light microscopes is in the order of microns, the two molecular species could be quite far apart in molecular terms and still appear to colocalize. In addition, because fluorophores are directly conjugated to the antibodies in conventional immunofluorescence, there is no way to amplify the signal in a manner analogous to the amplification that occurs when horse radish peroxidase or alkaline phosphatase conjugated antibodies are used. As a result, the sensitivity of the conventional immunofluorescence is limited.
New techniques which provide ways of assessing in vivo nucleic acid-protein, protein-protein, and other intermolecular interactions would be desirable, because they would provide new tools for studying gene regulation, enzymatic reactions, hormone-receptor interactions, and other cellular processes in vivo. In addition to the value that such new methods would bring to basic research, the ability to assess accurately in vivo nucleic acid-protein interactions could provide tools for isolating genes, identifying transcription factors, identifying regulatory regions, identifying transcription factor modulating agents and the like. The present invention provides, inter alia, new methods for assessing in vivo DNA-protein interactions, in vivo RNA-protein interactions, indirect protein-nucleic acid interactions and the like, thereby providing new tools for studying gene regulation, isolating genes, identifying transcription factors, identifying transcription factor modulating agents, identifying regulatory regions and many other features which will become apparent upon further reading. Also provided are new methods for visualizing other types of intermolecular interactions with high resolution and sensitivity.
The present invention provides methods which provide in vivo procedures for assessing protein-nucleic acid interactions. These methods represent a novel technology referred to generally as Protein Position Identification with Nuclease Tail or xe2x80x9cPINPOINTxe2x80x9d methods. In the methods, a fusion partner which binds directly or indirectly to a target nucleic acid is fused, typically via a linker, to an endonuclease domain which cleaves the target nucleic acid. By monitoring the in vivo cleavage of the target nucleic acid by the endonuclease domain, it is possible to quantitatively and qualitatively monitor interactions between proteins and nucleic acids, between protein complexes and nucleic acids, and between individual members of a protein complex and a nucleic acid. These methods are adapted to basic research, drug screening methods, methods of finding targets for transcription factors such as oncogenes, methods of finding transcription factors for target sequences, and the like.
The present invention further provides methods of screening test nucleic acids for in vivo binding sites which are cleaved by a chimeric guide endonuclease fusion molecule. Typically, a cell comprising a chimeric nucleic acid encoding the chimeric molecule and a test nucleic acid is provided, the chimeric nucleic acid is expressed in the cell, thereby producing a chimeric guide protein in the cell and, the cell is incubated under conditions in which the guide protein is active.
In one assay of the invention, the test nucleic acid includes a promoter sequence operably linked to a reporter gene. Detection of the presence or absence of reporter gene expression is an indicator for whether the test nucleic acid comprises an in vivo binding site for the chimeric guide molecule. In one class of embodiments, the cell is provided by co-transducing the cell with a plasmid encoding the target nucleic acid and a plasmid encoding a chimeric guide protein. Optionally, one or more additional plasmids comprising one or more additional test nucleic acids are also transduced into the cell, and the effect of the chimeric guide molecule is assessed simultaneously on more than one test nucleic acid. Parallel screening formats are provided, in which a second cell comprising a second chimeric nucleic acid encoding a second chimeric guide molecule and a second test nucleic acid is provided. The second chimeric nucleic acid is expressed and the effect of the chimeric nucleic acid on the second test nucleic acid is monitored.
The invention provides methods of detecting and assessing a nucleic acid binding molecule modulating agent. In the methods, a cell comprising a test nucleic acid binding site (e.g., a promoter sequence which is bound by a transcription factor) and a chimeric guide-endonuclease molecule is provided. The cell is contacted with the potential modulating agent, and the rate of cleavage of the test nucleic acid binding site by the chimeric molecule in the presence of the agent in measured. In one class of embodiments, the invention provides methods of cleaving target nucleic acids in vitro or in vivo. In the methods, a target nucleic acid is contacted by a guide-micrococcal endonuclease fusion molecule in the presence of calcium. The guide-micrococcal endonuclease fusion then cleaves the target nucleic acid. In one embodiment, the cleavage is performed in situ, e.g., in a tissue or cell sample on a solid substrate such as a microscope slide.
The present invention also provides methods for assessing intermolecular interactions both in vivo and in vitro. These methods represent a novel technology referred to generally as xe2x80x9cFLASHPOINTxe2x80x9d methods. In these methods, which are useful for detecting whether a first molecule is in close proximity to a second molecule, a molecular beacon is attached to the first molecule. The molecular beacon, which can be attached directly or indirectly to the first molecule, is typically an oligonucleotide to which is attached a fluorophore and a quencher. A chimeric endonuclease is attached to the second molecule, either directly or indirectly. To determine whether the first molecule is in close proximity to the second molecule, one detects whether fluorescence is emitted by the fluorophore.
Fluorescence emission is indicative of cleavage of the oligonucleotide by the endonuclease moiety, thereby causing separation of the fluorophore and the quencher. In a preferred embodiment, the endonuclease moiety is inducible (e.g., calcium inducible).
In another embodiment, the invention provides methods of obtaining increased sensitivity in assays such as immunoassays. The methods involve detecting a target molecule by contacting the target molecule with a chimeric endonuclease. The target molecule and the chimeric endonuclease are placed under conditions conducive to formation of an association between the target molecule and the chimeric endonuclease. The chimeric endonuclease is then contacted with a molecular beacon that is composed of an oligonucleotide to which is attached a fluorophore and a quencher. The presence or absence of a fluorescent signal is then detected. A signal, if present, results from cleavage of the oligonucleotide by the endonuclease, which causes separation of the quencher from the fluorophore.